US9701957B2 - Reagent holder, and kits containing same - Google Patents
Reagent holder, and kits containing same Download PDFInfo
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- US9701957B2 US9701957B2 US12/218,416 US21841608A US9701957B2 US 9701957 B2 US9701957 B2 US 9701957B2 US 21841608 A US21841608 A US 21841608A US 9701957 B2 US9701957 B2 US 9701957B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
- C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
- C08G83/002—Dendritic macromolecules
- C08G83/003—Dendrimers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
Definitions
- the technology described herein generally relates to holders for reagents, such as may be used for transporting the reagents and for carrying out processing operations with the reagents.
- the technology more particularly relates to holders that hold reagents for preparing biological samples for amplifying and detecting polynucleotides extracted from the samples.
- diagnostics industry is a critical element of today's healthcare infrastructure. At present, however, diagnostic analyses no matter how routine have become a bottleneck in patient care. There are several reasons for this. For example, many diagnostic analyses can only be done with highly specialist equipment that is both expensive and only operable by trained clinicians. Such equipment is found in only a few locations, and often there is just one in any given urban area. This means that most hospitals are required to send out samples for analyses to these locations, thereby incurring shipping costs and transportation delays, and possibly even sample loss or mishandling.
- a biological sample once extracted from a patient, must be put in a form suitable for a processing and detection regime that typically involves using PCR to amplify a vector of interest. Once amplified, the presence or absence of the vector in the sample needs to be determined unambiguously.
- Preparing samples for PCR is currently a time-consuming and labor intensive step, though not one requiring specialist skills, and could usefully be automated.
- steps such as PCR and nucleotide detection have customarily only been within the compass of specially trained individuals having access to specialist equipment.
- Sample preparation is labor intensive in part because of the number of reagents required, and the need for multiple liquid transfer (e.g., pipetting) operations. Furthermore, the reagents required are of sufficient variety that they typically require different handling from one another and are available from different vendors.
- a reagent holder comprising: a connecting member having an upper side and, opposed to the upper side, an underside; a process tube affixed to the connecting member and having an aperture located in the connecting member; at least one socket, located in the connecting member, the socket configured to accept a pipette tip; two or more reagent tubes disposed on the underside of the connecting member, each of the reagent tubes having an inlet aperture located in the connecting member; and one or more receptacles, located in the connecting member, wherein the one or more receptacles are each configured to receive a reagent tube inserted from the upper side of the connecting member.
- a kit comprising: a first pouch containing one or more of the holders described herein; and a second pouch, having an inert atmosphere inside, and one or more tubes containing lyophilized PCR reagents.
- a unitized reagent holder comprising: a strip, to which is attached: a single process tube; one or more containers, each of which holding a reagent selected from the group consisting of: a sample preparation reagent, PCR reagents for a first analyte, and one or more liquid reagents; and one or more sockets configured to hold one or more pipette tips.
- a reagent holder comprising: a process tube; one or more pipette tips; and one or more reagent tubes, wherein the one or more reagent tubes contain, respectively, sufficient quantities of one or more reagents for carrying out extraction of polynucleotides from a sample, and wherein the process tube, the one or more pipette tips, and the one or more reagent tubes are each joined to a single connecting member.
- a reagent tube comprising: a stellated pattern of cutouts or ridges centered at the bottom of the interior surface of the tube.
- FIGS. 1A and 1B show an first exemplary embodiment of a reagent holder having a pipette sheath, in perspective view ( FIG. 1A ) and underside view ( FIG. 1B ).
- FIG. 2 shows an exemplary embodiment of a reagent holder not having a pipette sheath, in perspective view.
- FIGS. 3A-3C show a second exemplary embodiment of a reagent holder having a pipette sheath, in perspective view ( FIG. 3A ) and cross-sectional view ( FIG. 3B ), and exploded view ( FIG. 3C ).
- FIGS. 4A and 4B show a stellated feature on the interior of a reagent tube, in cross-sectional ( FIG. 4A ) and plan ( FIG. 4B ) view.
- FIG. 5 shows a sequence of pipetting operations in conjunction with a reagent tube having a stellated feature.
- FIG. 6 shows embodiments of a laminated layer.
- FIG. 7 shows a sequence of pipetting operations in conjunction with a laminated layer.
- FIGS. 8A-8D shows an exemplary kit containing holders and reagents.
- FIG. 9 shows a rack for holding one or more holders.
- FIG. 10 shows a process tube heating unit, as used in conjunction with a rack.
- FIG. 11 shows a schematic of an automated apparatus configured to carry out sample preparation using a holder as described herein.
- reagent holders for holding and transporting reagents for various purposes, in particular sample preparation in a clinical context.
- the holder permits snapping in of one or more tubes of analyte specific reagents (ASR's) and/or sample preparation reagents, and carrying out liquid dispensing processes, associated with sample preparation, that minimize cross-sample contamination but permit multiple PCR preparations to be performed from a single clinical sample.
- ASR's analyte specific reagents
- the holders are configured for use in an automated preparatory apparatus that can carry out sample preparation on samples in more than one holder simultaneously.
- the reagent holders as described herein find particular application to analyzing any nucleic acid containing sample for any purpose, including but not limited to genetic testing, and clinical testing for various infectious diseases in humans.
- preparation of a PCR-ready sample for use in subsequent diagnosis can include one or more of the following steps: contacting a neutralized polynucleotide sample with a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides (in some embodiments, the PCR reagent mixture can further include a positive control plasmid and a fluorogenic hybridization probe selective for at least a portion of the plasmid). In some embodiments, the PCR reagent mixture can be in the form of one or more lyophilized pellets, as stored in a container on the holder, and the method can further include reconstituting the PCR pellet with liquid to create a PCR reagent mixture solution.
- the holder herein provides in a self-contained manner, all of the reagents required to prepare a PCR-ready sample, or, when delivered to a user in kit form, contains in conjunction with other packages all of the required reagents. Suitable reagents, and protocols for using the same in DNA and RNA extractions can be found in, respectively, copending application Ser. Nos. 12/172,208, and 12/172,214, both filed Jul. 11, 2008 and incorporated herein by reference.
- the holders herein are also configured for use by an apparatus that carries out automated sample preparation, for example, on multiple samples simultaneously.
- An exemplary form of such an apparatus is further described herein, and can also be found described in U.S. provisional patent application Ser. No. 60/959,437, filed Jul. 13, 2007, incorporated herein by reference in its entirety.
- FIGS. 1A and 1B show views of an exemplary holder 501 as further described herein.
- FIG. 2 shows a plan view of another exemplary holder 502 , as further described herein.
- FIG. 3A shows an exemplary holder 503 in perspective view
- FIG. 3B shows the same holder in cross-sectional view.
- FIG. 3C shows an exploded view of the same holder as in FIGS. 3A and 3B . All of these exemplary holders, as well as others consistent with the written description herein though not shown as specific embodiments, are now described.
- the exemplary holders shown in FIGS. 1A, 1B, 2, 3A, 3B, and 3C can each be referred to as a “unitized disposable strip”, or a “unitized strip”, because they are intended to be used as a single unit that is configured to hold all of the reagents and receptacles necessary to perform a sample preparation, and because they are laid out in a strip format. It is consistent with the description herein, though, that other geometric arrangements of the various receptacles are contemplated, so that the description is not limited to a linear, or strip, arrangement, but can include a circular or grid arrangement.
- reagents contained in the holder are provided as liquids, and others may be provided as solids.
- a different type of container or tube is used to store liquids from those that store the solids.
- the holder can be disposable, such as intended for a single use, following which it is discarded.
- the holder is typically made of a plastic such as polypropylene.
- the plastic is such that it has some flexibility to facilitate placement into a rack, as further described herein.
- the plastic is typically rigid, however, so that the holder will not significantly sag or flex under its own weight and will not easily deform during routine handling and transport, and thus will not permit reagents to leak out from it.
- the holder comprises a connecting member 510 having one or more characteristics as follows.
- Connecting member 510 serves to connect various components of the holder together.
- Connecting member 510 has an upper side 512 and, opposed to the upper side, an underside 514 .
- FIG. 1B a view of underside 514 is shown, having various struts 597 connecting a rim of the connecting member with variously the sockets, process tube, and reagent tubes.
- Struts 597 are optional, and may be omitted all or in part, or may be substituted by, in all or in part, other pieces that keep the holder together.
- the holder is configured to comprise: a process tube 520 affixed to the connecting member and having an aperture 522 located in the connecting member; at least one socket 530 , located in the connecting member, the socket configured to accept a disposable pipette tip 580 ; two or more reagent tubes 540 disposed on the underside of the connecting member, each of the reagent tubes having an inlet aperture 542 located in the connecting member; and one or more receptacles 550 , located in the connecting member, wherein the one or more receptacles are each configured to receive a complementary container such as a reagent tube (not shown) inserted from the upper side 512 of the connecting member.
- a complementary container such as a reagent tube (not shown) inserted from the upper side 512 of the connecting member.
- the holder is typically such that the connecting member, process tube, and the two or more reagent tubes are made from a single piece, such as a piece of polypropylene.
- the holder is also typically such that at least the process tube, and the two or more reagent tubes are translucent.
- the one or more receptacles 550 are configured to accept reagent tubes that contain, respectively, sufficient quantities of one or more reagents typically in solid form, such as in lyophilized form, for carrying out extraction of nucleic acid from a sample that is associated with the holder.
- the receptacles can be all of the same size and shape, or may be of different sizes and shapes from one another.
- Receptacles 550 are shown as having open bottoms, but are not limited to such topologies, and may be closed other than the inlet 552 in the upper side of connecting member 510 .
- the receptacles 550 are configured to accept commonly used containers in the field of laboratory analysis, or containers suitably configured for use with the holder herein.
- the containers are typically stored separately from the holders to facilitate sample handling, since solid reagents normally require different storage conditions from liquid reagents. In particular many solid reagents may be extremely moisture sensitive.
- the snapped-in reagent tubes containing different reagents may be of different colors, or color-coded for easy identification by the user.
- they may be made of different color material, such as tinted plastic, or may have some kind of identifying tag on them, such as a color stripe or dot. They may also have a label printed on the side, and/or may have an identifier such as a barcode on the sealing layer on the top.
- the containers 554 received by the receptacles 550 may alternatively be an integrated part of the holder and may be the same type of container as the waste chamber and/or the reagent tube(s), or may be different therefrom.
- the containers 554 containing lyophilized reagents, disposed in the receptacles 550 are 0.3 ml tubes that have been further configured to have a star pattern (see FIGS. 4A and 4B ) on their respective bottom interior surfaces. This is so that when a fluid has been added to the lyophilized reagents (which are dry in the initial package), a pipette tip can be bottomed out in the tube and still be able to withdraw almost the entire fluid from the tube, as shown in FIG. 5 , during the process of nucleic acid extraction.
- the design of the star-pattern is further described elsewhere herein.
- the reagent tubes such as containing the lyophilized reagents, can be sealed across their tops by a metal foil, such as an aluminum foil, with no plastic lining layer, as further described herein.
- Waste chamber 560 is optional and, in embodiments where it is present, is configured to receive spent liquid reagents. In other embodiments, where it is not present, spent liquid reagents can be transferred to and disposed of at a location outside of the holder, such as, for example, a sample tube that contained the original sample whose contents are being analyzed. Waste chamber 560 is shown as part of an assembly comprising additionally two or more reagent tubes 540 . It would be understood that such an arrangement is done for convenience, e.g., of manufacture; other locations of the waste chamber are possible, as are embodiments in which the waste chamber is adjacent a reagent tube, but not connected to it other than via the connecting member.
- the holder is typically such that the connecting member, process tube, the two or more reagent tubes, and the waste chamber (if present) are made from a single piece, made from a material such as polypropylene.
- the embodiments 501 and 503 are shown having a pipette sheath 570 .
- This is an optional component of the holders described herein. It may be permanently or removably affixed to connecting member 510 , or may be formed, e.g., moulded, as a part of a single piece assembly for the holder.
- exploded view of holder 503 in FIG. 3C shows lug-like attachments 574 on the upper surface of a removable pipette sheath 570 that engage with complementary recessed portions or holes in the underside 514 of connecting member 510 .
- Other configurations of attachment are possible.
- Pipette sheath 570 is typically configured to surround the at least one socket and a tip and lower portion of a pipette tip when the pipette tip is stationed in the at least one socket.
- the at least one socket comprises four sockets.
- the at least one socket comprises two, three, five, or six sockets.
- Pipette sheath 570 typically is configured to have a bottom 576 and a walled portion 578 disposed between the bottom and the connecting member. Pipette sheath 570 may additionally and optionally have one or more cut-out portions 572 in the wall 578 , or in the bottom 576 . Such cutouts provide ventilation for the pipette tips and also reduce the total amount of material used in manufacture of the holder.
- Embodiment 503 has a pipette sheath with no such cutouts.
- such a cutout is shown as an isosceles triangle in the upper portion of the sheath; a similar shaped cutout may be found at a corresponding position in the opposite side of the sheath, obscured from view in FIG. 1A .
- Other cutouts could have other triangular forms, circular, oval, square, rectangular, or other polygonal or irregular shapes, and be several, such as many, in number.
- the wall 578 of pipette sheath 570 may also have a mesh or frame like structure having fenestrations or interstices.
- a purpose of the sheath is to catch drips from used pipette tips, and thereby to prevent cross-sample contamination, from use of one holder to another in a similar location, and/or to any supporting rack in which the holder is situated.
- the bottom 576 is solid and bowl-shaped (concave) so that drips are retained within it.
- An embodiment such as 502 having no pipette sheath, could utilize, e.g., a drip tray or a drainage outlet, suitably placed beneath pipette tips located in the one or more sockets, for the same purpose.
- the pipette tip sheath prevents or inhibits the tips of other reagent holders—such as those that are situated adjacent to the one in question in a rack as further described herein—from touching each other when the tips are picked up and/or dropped off before or after some liquid processing step.
- Contact between tips in adjacent holders is generally not intended by, for example, an automated dispensing head that controls sample processing on holders in parallel, but the pipette tips being long can easily touch a tip in a nearby strip if the angle when dropping off of the tip deviates slightly from vertical.
- the holders of embodiments 501 , 502 , and 503 all have a connecting member that is configured so that the at least one socket, the one or more receptacles, and the respective apertures of the process tube, and the two or more reagent tubes, are all arranged linearly with respect to one another (i.e., their midpoints lie on the same axis).
- the holders herein are not limited to particular configurations of receptacles, waste chamber, process tube, sockets, and reagent tubes.
- a holder may be made shorter, if some apertures are staggered with respect to one another and occupy ‘off-axis’ positions.
- the various receptacles, etc. also do not need to occupy the same positions with respect to one another as is shown in FIGS. 3A and 3B , wherein the process tube is disposed approximately near the middle of the holder, liquid reagents are stored in receptacles mounted on one side of the process tube, and receptacles holding solid reagents are mounted on the other side of the process tube.
- the process tube is on one end of the connecting member, and the pipette sheath is at the other end, adjacent to, in an interior position, a waste chamber and two or more reagent tubes.
- Process tube 520 can also be a snap-in tube, rather than being part of an integrated piece. Process tube 520 is typically used for various mixing and reacting processes that occur during sample preparation. For example, cell lysis can occur in process tube 520 , as can extraction of nucleic acids. Process tube 520 is then advantageously positioned in a location that minimizes, overall, pipette head moving operations involved with transferring liquids to process tube 520 .
- Reagent tubes 540 are typically configured to hold liquid reagents, one per tube.
- Reagent tubes 540 are typically configured to hold liquid reagents, one per tube.
- three reagent tubes are shown, containing respectively wash buffer, release buffer, and neutralization buffer, each of which is used in a sample preparation protocol.
- Reagent tubes 540 that hold liquids or liquid reagents can be sealed with a laminate structure 598 .
- the laminate structure typically has a heat seal layer, a plastic layer such as a layer of polypropylene, and a layer of metal such as aluminum foil, wherein the heat seal layer is adjacent the one or more reagent tubes.
- the additional plastic film that is used in a laminate for receptacles that contain liquid reagents is typically to prevent liquid from contacting the aluminum.
- the heat seal layer 602 for example made of a laquer or other such polymer with a low melting point, is at the bottom, adjacent to the top of the holder, when so applied.
- the plastic layer 604 is typically on top of the heat seal layer, and is typically made of polypropylene, having a thickness in the range 10-50 microns.
- the metal layer 608 is typically on top of the plastic layer and may be a layer of Al foil bonded to the plastic layer with a layer of adhesive 606 , as in the first embodiment in FIG. 6 , or may be a layer of metal that is evaporated or sputtered into place directly on to the plastic layer.
- Exemplary thicknesses for the respective layers are shown in FIG. 6 , where it is to be understood that variations of up to a factor of 2 in thickness are consistent with the technology herein.
- the aluminum foil is 0.1-15 microns thick, and the polymer layer is 15-25 microns thick in one embodiment.
- the aluminum is 0.1-1 microns thick, and the polymer layer is 25-30 microns thick.
- the laminates deployed herein make longer term storage easier because the holder includes the presence of sealed lyophilized reagents as well as liquids sealed in close proximity, which is normally hard to achieve.
- the tops of the reagent tubes have beveled edges so that when an aluminum foil is heat bonded to the top, the plastic melt does not extend beyond the rim of the tube. This is advantageous because, if the plastic melt reduces the inner diameter of the tube, it will cause interference with the pipette tip during operation.
- a raised flat portion 599 facilitates application and removal of laminate 598 .
- Raised surface 599 , on the upper side of the connecting member, and surrounding the inlet apertures to the reagent tubes and, optionally, the waste chamber, is an optional feature of the holder.
- the manner in which liquid is pipetted out is such that a pipette tip piercing through the foil rips through without creating a seal around the pipette tip, as in FIG. 7 .
- Such a seal around the tip during pipetting would be disadvantageous because a certain amount of air flow is desirable for the pipetting operation.
- a seal is not created because the laminate structure causes the pierced foil to stay in the position initially adopted when it is pierced.
- the upper five panels in FIG. 7 illustrate the pipetting of a reagent out from a reagent tube sealed with a laminate as further described herein. At A, the pipette tip is positioned approximately centrally above the reagent tube that contains reagent 707 .
- the pipette tip is lowered, usually controllably lowered, into the reagent tube, and in so doing pierces the foil 598 .
- the exploded view of this area shows the edge of the pierced laminate to be in contact with the pipette tip at the widest portion at which it penetrates the reagent tube.
- the pipette tip is withdrawn slightly, maintaining the tip within the bulk of the reagent 707 .
- the exploded view shows that the pierced foil has retained the configuration that it adopted when it was pierced and the pipette tip descended to its deepest position within the reagent tube.
- the pipette tip sucks up reagent 707 , possibly altering its height as more and more older people undergo such tests.
- the pipette tip is removed entirely from the reagent tube.
- the materials of the various tubes and chambers may be configured to have at least an interior surface smoothness and surface coating to reduce binding of DNA and other macromolecules thereto. Binding of DNA is unwanted because of the reduced sensitivity that is likely to result in subsequent detection and analysis of the DNA that is not trapped on the surface of the holder.
- the process tube also may have a low binding surface, and allows magnetic beads to slide up and down the inside wall easily without sticking to it. Moreover, it has a hydrophobic surface coating enabling low stiction of fluid and hence low binding of nucleic acids and other molecules.
- the holder comprises a registration member such as a mechanical key.
- a mechanical key ensures that the holder is accepted by a complementary member in, for example, a supporting rack or a receiving bay of an apparatus that controls pipetting operations on reagents in the holder.
- a mechanical key is normally a particular-shaped cut-out that matches a corresponding cutout or protrusion in a receiving apparatus.
- embodiment 501 has a mechanical key 592 that comprises a pair of rectangular-shaped cut-outs on one end of the connecting member. This feature as shown additionally provides for a tab by which a user may gain a suitable purchase when inserting and removing the holder into a rack or another apparatus.
- Embodiments 501 and 502 also have a mechanical key 590 at the other end of connecting member 510 .
- Key 590 is an angled cutout that eases insertion of the holder into a rack, as well as ensures a good registration therein when abutting a complementary angled cut out in a recessed area configured to receive the holder.
- Other variations of a mechanical key are, of course, consistent with the description herein: for example, curved cutouts, or various combinations of notches or protrusions all would facilitate secure registration of the holder.
- the holder further comprises an identifier affixed to the connecting member.
- the identifier may be a label, such as a writable label, a bar-code, a 2-dimensional bar-code, or an RFID tag.
- the identifier can be, e.g., for the purpose of revealing quickly what combination of reagents is present in the holder and, thus, for what type of sample preparation protocol it is intended.
- the identifier may also indicate the batch from which the holder was made, for quality control or record-keeping purposes.
- the identifier may also permit a user to match a particular holder with a particular sample.
- a holder additionally can be configured to accept a sample, such as in a sample tube.
- a rack accepts a number of sample tubes and a number of corresponding holders in such a manner that the sample tubes and holders can be separately and independently loaded from one another.
- a holder can be configured to also accept a sample, for example in a sample tube.
- a complementary rack is configured to accept a number of holders, wherein each holder has a sample as well as reagents and other items.
- the holder is configured so that the sample is accessible to a sample identification verifier.
- the holder described herein may be provided in a sealed pouch, to reduce the chance of air and moisture coming into contact with the reagents in the holder.
- a sealed pouch may contain one or more of the holders described herein, such as 2, 4, 6, 8, 10, 12, 16, 20, or 24 holders.
- the holder may also be provided as part of a kit for carrying out sample preparation, wherein the kit comprises a first pouch containing one or more of the holders described herein, each of the holders configured with liquid reagents for, e.g., lysis, wash, and release, and a second pouch, having an inert atmosphere inside, and one or more reagent tubes containing lyophilized PCR reagents, as shown in FIGS. 8A-D .
- the kit comprises a first pouch containing one or more of the holders described herein, each of the holders configured with liquid reagents for, e.g., lysis, wash, and release, and a second pouch, having an inert atmosphere inside, and one or more reagent tubes containing lyophilized PCR reagents, as shown in FIGS. 8A-D .
- kit may also be configured to provide for analysis of multiple samples, and contain sufficient PCR reagents (or other amplification reagents, such as for RT-PCR, transcription mediated amplification, strand displacement amplification, NASBA, helicase dependent amplification, and other familiar to one of ordinary skill in the art, and others described herein) to process such samples, and a number of individual holders such as 2, 4, 6, 8, 10, 12, 16, 20, or 24 holders.
- PCR reagents or other amplification reagents, such as for RT-PCR, transcription mediated amplification, strand displacement amplification, NASBA, helicase dependent amplification, and other familiar to one of ordinary skill in the art, and others described herein
- the containers 554 that contain lyophilized reagents are 0.3 ml tubes that have been further configured to have a star-shaped—or stellated—pattern (see FIGS. 4A and 4B ) on their respective bottom interior surfaces.
- Still other tubes for use herein, as well as for other uses not herein described, can be similarly configured.
- the benefits afforded by the star-shaped pattern also accrue to reagent tubes that contain liquid samples that are directly pipetted out of the tubes (as well as to those tubes that initially hold solids that are constituted into liquid form prior to pipetting).
- Other size tubes that would benefit from such a star-shaped pattern have sizes in the range 0.1 ml to 0.65 ml. for example.
- the star-shaped pattern ensures that when a fluid is withdrawn from the tube, a pipette tip can be bottomed out in the tube and still be able to withdraw the entire, or almost the entire fluid from the tube, as shown in FIG. 5 . This is important because, when working with such small volumes, and when target DNA can be present in very few copies, sample loss due to imperfections of pipetting is to be minimized to every extent possible.
- the design of the star shaped pattern is important, especially when using for recovery of DNA/RNA present in very small numbers in the clinical sample.
- the stellated pattern should enable pipetting of most of the liquid (residual volume ⁇ 1 microliter) when used with a pipette bottomed out with the bottom of the tube. Additionally, the stellated pattern should be designed to minimize surface area as well as dead-end grooves that tend to have two undesirable effects—to trap liquid as well as to increase undesirable retention of polynucleotides by adsorption.
- FIG. 5 has a number of panels, A-G, each representing, in sequence, a stage in a pipetting operation.
- a pipette tip 2210 containing a liquid 2211 (such as a buffer solution), is positioned directly or approximately above the center of reagent tube 2200 .
- the tube contains a number of lyophilized pellets 2212 , and is sealed by a layer 2214 , such as of foil.
- the foil may be heat-sealed on to the top of the tube.
- the top of the reagent tube has chamfer edges to reduce expansion of the top rim of the tube during heat sealing of a foil on the top of the tube.
- the tube may further comprise an identifiable code, such as a 1-D or a 2-D bar-code on the top. Such a code is useful for identifying the composition of the reagents stored within, and/or a batch number for the preparation thereof, and/or an expiry date.
- the code may be printed on with, for example, an inkjet or transfer printer.
- Stellated pattern 2203 on the bottom interior surface of the tube 2200 is shown.
- the pipette tip is lowered, piercing seal 2214 , and brought into a position above the particles 2212 .
- the liquid 2211 is discharged from the pipette tip on to the particles, dissolving the same, as shown at D.
- the pipette tip is lowered to a position where it is in contact with the stellated pattern 2203 .
- a E the pipette tip is caused to suck up the solution 2218 , and at F, the tip may optionally discharge the solution back into the tube.
- Steps E and F may be repeated, as desired, to facilitate dissolution and mixing of the lyophilized components into solution.
- step G after sucking up as much of the solution 2218 as is practicable into the pipette tip, the pipette tip is withdrawn from the tube. Ideally, 100% by volume of the solution 2218 is drawn up into the pipette tip at G. In other embodiments, and depending upon the nature of solution 2218 , at least 99% by volume of the solution is drawn up. In still other embodiments, at least 98%, at least 97%, at least 96%, at least 95%, and at least 90% by volume of the solution is drawn up.
- the design of the stellated or star-shaped pattern can be optimized to maximize the flow rate of liquid through the gaps in-between a bottomed out pipette, such as a p1000 pipette, and the star pattern, and is further described in U.S. provisional patent application Ser. No. 60/959,437, filed Jul. 13, 2007, incorporated herein by reference. It would be understood that, although the description herein pertains to pipettes and pipette tips typically used in sample preparation of biological samples, the principles and detailed aspects of the design are as applicable to other types of pipette and pipette tip, and may be so-adapted.
- FIG. 4A shows a cross sectional perspective view of a reagent tube 2200 having side wall 2201 and bottom 2202 . Interior surface 2204 of the bottom is visible. A star-shaped cutout 2203 is shown in part, as three apical grooves.
- the star-shaped pattern is present as a raised portion on the lower interior surface of the tube.
- an outer portion of the mould is a cavity defining the exterior shape of the tube.
- An interior shape of the tube is formed by a mould positioned concentrically with the outer portion mould, and having a star-shaped structure milled out of its tip.
- the star-shape is formed as a raised portion on the bottom interior surface of the tube.
- the exemplary star pattern 2203 shown in FIG. 4B in plan view resembles a “ship's wheel” and comprises a center 2209 , a circular ring 2207 centered on center 2209 , and 8 radial segments configured as radial grooves 2205 .
- Each groove meets the other grooves at center 2209 , and has a radial end, also referred to as an apex or vertex.
- Star pattern 2203 has 8 grooves, but it would be understood that a star pattern having fewer or a greater number of grooves, such as 3, 4, 6, 10, or 12, would be consistent with the design herein.
- the number of grooves of the star should be minimum consistent with effective liquid pipetting and also spaced apart enough not to trap the tip of any of the pipette tips to be used in the liquid handling applications.
- Center 2209 is typically positioned coincidentally with the geometric center of the bottom of reagent tube 2200 .
- the tube is typically circular in cross-section, so identifying its center (e.g., at a crossing point of two diameters) is normally straightforward.
- Center 2209 may be larger than shown in FIG. 4B , such as may be a circular cutout or raised portion that exceeds in diameter of the region formed by the meeting point of grooves 2205 .
- Ring 2207 is an optional feature of star-shaped pattern 2203 .
- ring 2207 is centered about center 2209 , and typically it also has a dimension that corresponds to the lower surface of a pipette tip.
- Ring 2207 is thus preferably a cut-our or recessed feature that can accommodate the pipette tip and assist in guiding its positioning centrally at the bottom of the tube. In other embodiments more than one, such as 2, 3, or 4 concentric rings 2207 are present.
- the star pattern is configured to have dimensions that give an optimal flow-rate of liquid out of the reagent tube into a suitably positioned pipette tip.
- the star pattern is shown in FIG. 4B as being significantly smaller in diameter than the diameter of the tube at its widest point.
- the star pattern may have, in various embodiments, a diameter (measured from center 2209 to apex of a groove 2205 ) from 5-20% of the diameter of the reagent tube, or from 10-25% of the diameter of the reagent tube, or from 15-30% of the diameter of the reagent tube, or from 20-40% of the diameter of the reagent tube, or from 25-50% of the diameter of the reagent tube, or from 30-50% the diameter of the reagent tube, or from 40-60% the diameter of the reagent tube, or from 50-75% the diameter of the reagent tube, or from 65-90% the diameter of the reagent tube.
- the grooves 2205 are thus separated by ridges (occupying the space in between adjacent grooves).
- the grooves are narrower (occupy a smaller radial angle) than the gaps between them.
- the grooves may be proportionately wider than the gaps between them. In such embodiments, it may be more appropriate to describe them as having ridges instead of grooves.
- the grooves and ridges that separate them are of equal widths at each radial distance from the center.
- the grooves that form the apices of the star may be rounded in their lower surfaces, such as semi-circular in cross section, but are typically V-shaped. They may also be trapezoid in cross-section, such as having a wider upper portion than the bottom, which is flat, the upper portion and the bottom being connected by sloping walls.
- the grooves end on the same level in the bottom of the tube.
- the radial ends are all disposed on the circumference of a circle.
- the grooves do not all end on the same level.
- grooves may alternately end on different levels, and thus the ends are alternately disposed on the respective circumferences of two circles that occupy different planes in space from one another.
- Grooves 2205 are shown in FIG. 4B as having equal lengths (as measured from center 2209 to apex). This need not be so. In alternative embodiments, grooves may have different lengths from one another, for example, as alternating lengths on alternating grooves, where there are an even number of grooves. Furthermore, apices may be rounded, rather than pointed.
- the grooves taper uniformly in width and depth from center 2209 to each respective apex.
- Still other configurations are possible, such as a groove that follows a constant width, or depth, out to a particular radial extent, such as 30-60% of its length, and then narrows or becomes shallower towards its apex.
- a groove may start narrow at center 2209 , widen to a widest region near its midpoint of length, and then narrow towards its apex. Still other possibilities, not described herein, are consistent with the stellated pattern.
- each groove 2205 at its widest point is typically around 50 microns, and the width typically tapers uniformly from a widest point, closest to or at center 2209 , to the apex.
- the depth of a groove at the deepest point is typically around 25-50 microns and the depth typically tapers uniformly from a deepest point, closest to or at center 2209 , to an apex.
- the radius of the star formed from the grooves is typically around 0.5 mm, but may be from 0.1-1 mm, or from 0.3-2 mm.
- the grooves in a 0.3 ml tube, should be rounded off and less than 100 microns deep, or less than 50 microns deep, or less than 25 microns deep.
- the stellated pattern typically has a rotation axis of symmetry, the axis disposed perpendicular to the bottom of the tube and through center 2209 , so that the grooves are disposed symmetrically about the rotation axis.
- a rotation of 2 ⁇ /n about the central (rotational) axis can bring each groove into coincidence with the groove adjacent to it.
- the stellated shape shown in FIG. 4B is not limiting in that it comprises a number of radially disposed grooves 2205 , and an optional circular ring 2207 .
- Other star-shaped geometries may be used, and, depending upon ease of manufacture, may be preferred.
- a star can be created simply be superimposing two or more polygons having a common center, but offset rotationally with respect to one another about the central axis. (See, for example “star polygons” described at http://mathworld.wolfram.com/StarPolygon.html.) Such alternative manners of creating star-shaped patterns are utilizable herein.
- the holders herein are typically configured to fit into a dedicated rack, the rack being configured to hold one or more such holders, either on a laboratory benchtop, or situated in a dedicated apparatus having one or more other functions, such as automated pipetting.
- Two perspective views of an exemplary rack 800 configured to accept 12 sample tubes and 12 holders, are shown in FIG. 9 .
- a rack may accept 2, 4, 6, 8, 10, 12, 16, or 20 samples, in sample tubes 802 , and corresponding holders 804 .
- the holders each slide into, and lock into, place in the rack, such as with a cam locking mechanism that is recognized as locked audibly and/or physically, or such as with a mechanical key.
- the rack can be configured so that the holders, when positioned in it, are aligned for proper pipette tip pick-up using an automated liquid dispenser as further described herein.
- the rack is configured to accept the samples in individual sample tubes 802 , each mounted adjacent to a corresponding holder.
- the rack can be designed so that it can be easily removed from a dedicated apparatus and carried between the laboratory environment external to the apparatus, such as a bench, and the apparatus.
- the rack is designed to be stable on a horizontal surface, and not easily toppled over during carriage.
- the rack has a handle 806 to ease lifting and moving, and the handle can be locked into a vertical position, during carriage, also to reduce risk of the rack being toppled over.
- the handle has a soft grip 808 in its middle, and is attached to a metallic base member 810 having 4 feet that also serve as position locators when inserting the rack into a dedicated apparatus.
- the portion of the rack 812 that accepts the samples and holders can be made of plastic, and comprises 12 slots, and may be disposable.
- the interior of the rack around the location of process tubes in the various holders is configured to have clearance for a heater assembly and/or a magnetic separator as further described herein.
- the rack is configured so that process tubes on the individual holders are accepted by heater units in a heater assembly as further described in U.S. provisional patent application Ser. No. 60/959,437, filed Jul. 13, 2007.
- the holders herein are configured to accept pipette operations both performed manually by an operator, and by an automated pipette head.
- An exemplary automated pipette head is described in U.S. provisional application Ser. No. 60/959,437, filed Jul. 13, 2007, and incorporated herein by reference in its entirety.
- Typical features of an automated pipette head suitable for operating with holders as described herein include at least: an ability to pick up pipette tips from the one or more sockets, and to return pipette tips to such sockets after use; to strip and discard a pipette tip from a pipette head after use or upon encountering an error; move a pipette tip with precision from one location of a given holder to another so that, for example, liquid reagents can be located and added to solid reagents to make up solutions, and various liquid reagents can be mixed with one another during a sample preparation protocol.
- it is desirable that such an automated pipette device can operate on several, such as 2, 3, 4, or 6, holders simultaneously, and thereby perform certain operations in parallel.
- the pipette head should move in three degrees of freedom.
- the holders herein are configured so that the process tube is heated by a dedicated heating unit 1001 , for example situated in an apparatus for carrying out sample preparation on multiple samples in parallel, as shown in FIG. 10 .
- a heater assembly comprises one or more independently controllable heater units 1010 , each of which comprises a heat block configured to heat a process tube in a holder.
- a heat element is a power resistor.
- the right hand panel of FIG. 10 shows how holders loaded in a rack can be positioned in close proximity to a dedicated heating unit.
- the holders herein are configured so that the process tube is in close enough proximity to a magnetic assembly that separation of magnetic particles from reagents in solution in the process tube can be accomplished.
- An exemplary magnetic separator is configured to move one or more magnets relative to the one or more process tubes.
- the magnet is mounted in such a way that it can be moved in proximity to the process tubes, either in an automated fashion such as under control of a processor, or manually.
- the magnet can be made of neodymium (e.g., from K &J Magnetics, Inc.) and can have a magnetic strength of 5,000-15000 Gauss (Brmax).
- the poles of the magnets can be arranged such that one pole faces the heat blocks and the other faces away from the heat blocks.
- the heater assembly and magnetic separator operate together to permit successive heating and separation operations to be performed on liquid materials in the one or more process tubes without transporting either the liquid materials or the process tubes to different locations to perform either heating or separation.
- An exemplary heater assembly and magnetic separator are further described in U.S. provisional patent application Ser. No. 60/959,437, filed Jul. 13, 2007, and incorporated herein by reference in its entirety.
- FIG. 11 A schematic overview of an apparatus 981 for carrying out automated sample preparation on multiple samples in parallel, according to steps exemplified elsewhere herein, is shown in FIG. 11 .
- the geometric arrangement of the components of system 981 is exemplary and not intended to be limiting.
- a processor 980 such as a microprocessor, is configured to control functions of various components of the system as shown, and is thereby in communication with each such component requiring control. It is to be understood that many such control functions can optionally be carried out manually, and not under control of the processor. Furthermore, the order in which the various functions are described, in the following, is not limiting upon the order in which the processor executes instructions when the apparatus is operating. Thus, processor 980 can be configured to receive data about a sample to be analyzed, e.g., from a sample reader 990 , which may be a barcode reader, an optical character reader, or an RFID scanner (radio frequency tag reader).
- a sample reader 990 which may be a barcode reader, an optical character reader, or an RFID scanner (radio frequency tag reader).
- Processor 980 can be configured to accept user instructions from an input device 984 , where such instructions may include instructions to start analyzing the sample, and choices of operating conditions.
- Processor 980 can be also configured to communicate with a display 982 , so that, for example, information about an analysis is transmitted to the display and thereby communicated to a user of the system. Such information includes but is not limited to: the current status of the apparatus; progress of PCR thermocycling; and a warning message in case of malfunction of either system or cartridge.
- processor 980 may transmit one or more questions to be displayed on display 982 that prompt a user to provide input in response thereto.
- input 984 and display 982 are integrated with one another.
- Processor 980 can be optionally further configured to transmit results of an analysis to an output device 986 such as a printer, a visual display, a display that utilizes a holographic projection, or a speaker, or a combination thereof.
- Processor 980 can be still further optionally connected via a communication interface such as a network interface to a computer network 988 .
- Processor 980 can be further configured to control various aspects of sample preparation and diagnosis, as follows in overview.
- the apparatus 981 is configured to operate in conjunction with a complementary rack 970 .
- the rack is itself configured, as further described herein, to receive a number of biological samples 996 in a form suitable for work-up and diagnostic analysis, and a number of holders 972 —as further described herein, such as in connection with FIGS. 1A, 1B, 2, and 3A -C, that are equipped with various reagents, pipette tips and receptacles.
- the rack is configured so that, during sample work-up, samples are processed in the respective holders, the processing including being subjected, individually, to heating and cooling via heater assembly 977 .
- Heater assembly 977 operates in conjunction with a separator 978 , such as a magnetic separator, that also can be controlled by processor 980 to move into and out of close proximity to one or more processing chambers associated with the holders 972 , wherein particles such as magnetic particles are present.
- a separator 978 such as a magnetic separator
- Liquid dispenser 976 which similarly can be controlled by processor 980 , is configured to carry out various suck and dispense operations on respective sample, fluids and reagents in the holders 972 , to achieve extraction of nucleic acid from the samples. Liquid dispenser 976 can carry out such operations on multiple holders simultaneously.
- Sample reader 990 is configured to transmit identifying indicia about the sample, and in some instances the holder, to processor 980 .
- a sample reader is attached to the liquid dispenser and can thereby read indicia about a sample above which the liquid dispenser is situated.
- the sample reader is not attached to the liquid dispenser and is independently movable, under control of the processor.
- Liquid dispenser 976 is also configured to take aliquots of fluid containing nucleic acid extracted from one or more samples and direct them to storage area 974 , which may be a cooler.
- Area 974 contains, for example, a PCR tube corresponding to each sample.
- Embodiments of the apparatus shown in outline in FIG. 11 are advantageous because they do not require locations within the apparatus suitably configured for storage of reagents. Therefore, the apparatus in FIG. 11 is self-contained and operates in conjunction with holders 972 , wherein the holders are pre-packaged with reagents, such as in locations within it dedicated to reagent storage.
- the apparatus of FIG. 11 may be configured to carry out operation in a single location, such as a laboratory setting, or may be portable so that they can accompany, e.g., a physician, or other healthcare professional, who may visit patients at different locations.
- the apparatus is typically provided with a power-cord so that they can accept AC power from a mains supply or generator.
- the apparatus may also be configured to operate by using one or more batteries and therefore is also typically equipped with a battery recharging system, and various warning devices that alert a user if battery power is becoming too low to reliably initiate or complete a diagnostic analysis.
- the apparatus of FIG. 11 may further be configured, in other embodiments, for multiplexed sample analysis and/or analysis of multiple batches of samples, where, e.g., a single rack holds a single batch of samples.
- Each component shown in FIG. 11 may therefore be present as many times as there are batches of samples, though the various components may be configured in a common housing.
- An exemplary reagent holder consistent with the description herein has the following dimensions and capacities:
- these dimensions are exemplary. However, it is particularly desirable to ensure that a holder does not exceed these dimensions so that a rack and an apparatus that accommodates the reagent holder(s) does not become inconveniently large, and can be suitably situated in a laboratory, e.g., on a bench-top.
- Simple fixtures can be designed and machined to enable handling and processing of multiple strips. There are five steps that can be performed to produce this component.
- the disposable reagent holder will be placed in a fixture and filled with liquids using manual/electric-multiple pipetting.
- foil will be heat sealed to the plastic using exemplary heat seal equipment (Hix FH-3000-D Flat Head Press) and the foil trimmed as required.
- all pellets in tubes can be snapped into the strip, pipette tips can be inserted in their respective sockets, and a barcode label can be affixed.
- Desiccant packs can be placed into the blow molded or thermoformed rack designed to house 12 holders. Twelve disposable strips will be loaded into the rack and then sealed with foil. The sealed bag will be placed into a carton and labeled for shipping.
- Tubes containing buffers have to be sealed with high moisture vapor barrier materials in order to retain the liquid over a long period of time.
- Disposable holders may need to have a shelf life of 1-2 years, and as such, they should not lose more than say 10-15% of the liquid volume over the time period, to maintain required volume of liquid, and to maintain the concentration of various molecules present in the solution.
- the materials used for construction of the tube as well as the sealing laminate should not react with the liquid buffer.
- Special plastic laminates may provide the moisture barrier but they may have to be very thick (more than 300 ⁇ m thick), causing the piercing force to go up tremendously, or of special, expensive polymer (such as Aclar).
- Aluminum foils even a thin foil of a few hundred angstrom provides an effective moisture barrier but bare aluminum reacts with some liquid buffers, such as sodium hydroxide, even an aluminum foil with a sprayed coating of a non-reactive polymer may not be able to withstand the corrosive vapors over a long time. They may react through tiny pin holes present in the coating and may fail as a barrier over time.
- the aluminum laminate containing a plastic film described elsewhere herein serves well for not reacting with corrosive reagents such as buffers containing NaOH, and having the favorable properties of pierceability and acting as a moisture barrier.
- corrosive reagents such as buffers containing NaOH
- the aluminum foil tends to burst into an irregular polygonal pattern bigger than the diameter of the pipette
- the plastic film tends to wrap around the pipette tip with minimal gap between the pipette and the plastic film.
- the diameter of the hole in the plastic film is similar to the maximum diameter of the pipette that had crossed through the laminate. This wrapping of the pipette causes difficulty in dispensing and pipetting operations unless there is a vent hole allowing pressures to equilibrate between outside of the tube and the air inside of the tube.
- a strategy for successful pipetting of fluid is as follows:
- the containers of lyophilized reagents provided in conjunction with a holder as described herein are typically sealed by a non-plasticized aluminum foil (i.e., not a laminate as is used to seal the reagent tubes).
- Aluminum foil bursts into an irregular polygonal pattern when pierced through a pipette and leaves an air vent even though the pipette is moved to the bottom of the tube.
- a star-ridged (stellated) pattern is placed at the bottom of the container to maximize liquid volume withdrawn, and flow velocity in between the ridges.
- Exemplary steps for dissolving and withdrawing fluid are as follows:
- the material, surface properties, surface finish has a profound impact on the sensitivity of the assay performed.
- DNA/RNA as low as 50 copies/sample ( ⁇ 1 ml volume) need to be positively detected in a background of billions of other molecules, some of which strongly inhibit PCR.
- the surface of the reaction tube as well as the material of the surface has to be chosen to have minimal binding of polynucleotides.
- the inherent surfaces created by machining may have large surface area due to cutting marks as large as tens of microns of peaks and valleys.
Abstract
Description
-
- 180 mm long×22 mm wide×100 mm tall;
- Made from Polypropylene.
- One snapped-in low binding 1.7 ml tube that functions as a process tube.
- 3 built-in tubes that function as receptacles for reagents, as follows:
- One tube containing 200-1000 μl of wash buffer (0.1 mM Tris, pH 8).
- One tube containing 200-1000 μl of release solution (40 mM NaOH).
- One tube containing 200-1000 μl of neutralization solution (330 mM Tris, pH 8.0).
- One built-in tube that functions as a waste chamber (will hold ˜4 ml of liquid waste).
- 3 receptacles to accept containers for solid reagents. Snap-in 0.3 ml or 0.65 ml PCR tubes (which are typically stored separately from the reagent holder) are placed in each of these locations, and contain, respectively:
- lyophilized sample preparation reagents (lysis enzyme mix and magnetic affinity beads).
- First lyophilized PCR master mix, probes and primers for a first target analyte detection.
- Second lyophilized PCR master mix, probes and primers for a second target analyte detection (only offered in select cases, such as detection of Chlamydia and Gonorrhea from urine).
- 4 pipette tips located in 4 respective sockets.
- Pipette tip Sheath: The pipette tips have a sheath/drip tray underneath to help capture any drip from the pipette tips after being used, and also to prevent unwanted contamination of the instrument.
- Handle and Flex-Lock allows easy insertion, removal, and positive location of strip in rack.
- One or more labels: positioned upward facing to facilitate ease of reading by eye and/or, e.g., a bar-code reader, the one or more labels containing human and machine readable information pertaining to the analysis to be performed.
-
- Heat seals to unitized polypropylene strip (sealing temp ˜170-180° C.)
- No wrinkling, cracking and crazing of the foil after sealing
-
- Loss of less than 10% liquid (20 microliters from a volume of 200 microliter) for a period of 1 year stored at ambient temperature and pressure. (effective area of transport is ˜63 mm2); Approximate MVTR 0.8 cc/m2/day
-
- Ability to not react with 40 mM Sodium Hydroxide (pH<12.6): foil should have a plastic laminate at least 15 microns thick closer to the sealed fluid.
- Ability to not react with other buffers containing mild detergents
-
- Ability to puncture using a p1000 pipette with a force less than 3 lb
- Before puncturing, a fully supported membrane 8 mm in diameter will not stretch more than 5 mm in the orthogonal direction
- After puncturing, the foil should not seal the pipette tip around the circumference of the pipette.
-
- Pin-hole free
- No bubbles in case of multi-laminate structures.
-
- 1. Pierce through the laminate structure and have the pipette go close to the bottom of the reagent tube so that the hole created in the laminate is almost as big as the maximum diameter of the pipette (e.g., ˜6 mm for a p1000 pipette)
- 2. Withdraw the pipette up a short distance so that a small annular vent hole is left between the pipette and the laminate. The p1000 pipette has a smallest outer diameter of 1 mm and maximum outer diameter of 6 mm and the conical section of the pipette is about 28 mm long. A vent hole thickness of a hundred microns is enough to create a reliable vent hole. This corresponds to the pipette inserted to a diameter of 5.8 mm, leaving an annulus of 0.1 mm around it.
- 3. Withdraw fluid from the tube. Note that the tube is designed to hold more fluid than is necessary to withdraw from it for a sample preparation procedure.
-
- 1. Pierce through the pipette and dispense the fluid away from the lyophilized material. If the pipette goes below the level of the lyophilized material, it will go into the pipette and may cause jamming of the liquid flow out of the pipette.
- 2. Let the lyophilized material dissolve for a few seconds.
- 3. Move pipette down touching the ridged-bottom of the tube
- 4. Perform an adequate number of suck and spit operations (4-10) to thoroughly mix the reagents with the liquid buffer.
- 5. Withdraw all the reagents and move pipette to dispense it into the next processing tube.
Claims (9)
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US12/904,901 US8216530B2 (en) | 2007-07-13 | 2010-10-14 | Reagent tube |
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US13/692,980 Active US8710211B2 (en) | 2007-07-13 | 2012-12-03 | Polynucleotide capture materials, and methods of using same |
US14/262,525 Active US9217143B2 (en) | 2007-07-13 | 2014-04-25 | Polynucleotide capture materials, and methods of using same |
US14/976,746 Active 2028-12-25 US10100302B2 (en) | 2007-07-13 | 2015-12-21 | Polynucleotide capture materials, and methods of using same |
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USD814652S1 (en) * | 2016-10-14 | 2018-04-03 | Spartan Bioscience In. | Cartridge |
USD824041S1 (en) * | 2016-01-12 | 2018-07-24 | Universal Bio Research Co., Ltd. | Photometric reaction container |
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